Computational repurposing and preclinical validation of colquhounia root tablets for membranous nephropathy

Dear Editor, Current therapeutics of membranous nephropathy (MN) hold dilemmas of limited clinical response rates, high recurrence rate, and unavoidable adverse effects.1 Colquhounia Root Tablet (CRT) is a Chinese patent medicine prepared from the peeled root of Tripterygium hypoglaucum (Lévl.) Hutch (SFDA approval number: Z20027411). Emerging clinical evidence has indicated a potential therapeutic effect of CRT on MN.2,3 However, the mechanisms of CRT against MN remain unclear. In this study, 585 putative targets of CRT were predicted hit by chemical compounds identified by UPLC-Q-TOFMS (Figure S1 and Table S1) and collected from literatures (Table S2).4,5 Then, 692 MN-related genes were collected by searching clinical symptoms and the related genes (Table S3). After constructing the ‘disease-related genesdrug putative targets’ interaction network using the links among MN-related genes and CRT putative targets, a total of 49 key network targets were selected by calculating nodes’ topological features (Table S4). Functionally, the key putative targets of CRT against MN were significantly enriched in 11 signalling pathways, including JAK-STAT signalling, TNF signalling, PI3K-Akt signalling and etc. (all p < .001, Figure 1A). In the most enriched ‘immunization’ module, tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and their downstream partners (JAK2, STAT3, MMP9) formed a signal axis that may play an essential role in the pathogenesis of MN. Thus, we hypothesized that CRT might alleviate the severity of MN through regulating TNF-α-IL-6-JAK2/STAT3-MMP9 signalling axis (Figure 1B,C). To evaluate the effects of CRT on cationic bovine serum albumin (C-BSA) induced MN mice, kidney index, levels of microalbumin from 24 h urine, creatinine, total cholesterol and serum albumin were detected after the routine induction of MN mice and drug treatment (Figure 2A). The microalbumin levels from 24 h urine were significantly increased in MN mice from the 4th week till the end of the experiment (p < .001, Figure 2B). MN mice

Dear Editor, Current therapeutics of membranous nephropathy (MN) hold dilemmas of limited clinical response rates, high recurrence rate, and unavoidable adverse effects. 1 Colquhounia Root Tablet (CRT) is a Chinese patent medicine prepared from the peeled root of Tripterygium hypoglaucum (Lévl.) Hutch (SFDA approval number: Z20027411). Emerging clinical evidence has indicated a potential therapeutic effect of CRT on MN. 2,3 However, the mechanisms of CRT against MN remain unclear.
In this study, 585 putative targets of CRT were predicted hit by chemical compounds identified by UPLC-Q-TOF-MS ( Figure S1 and Table S1) and collected from literatures (Table S2). 4,5 Then, 692 MN-related genes were collected by searching clinical symptoms and the related genes (Table S3). After constructing the 'disease-related genesdrug putative targets' interaction network using the links among MN-related genes and CRT putative targets, a total of 49 key network targets were selected by calculating nodes' topological features (Table S4). Functionally, the key putative targets of CRT against MN were significantly enriched in 11 signalling pathways, including JAK-STAT signalling, TNF signalling, PI3K-Akt signalling and etc. (all p < .001, Figure 1A). In the most enriched 'immunization' module, tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and their downstream partners (JAK2, STAT3, MMP9) formed a signal axis that may play an essential role in the pathogenesis of MN. Thus, we hypothesized that CRT might alleviate the severity of MN through regulating TNF-α-IL-6-JAK2/STAT3-MMP9 signalling axis ( Figure 1B,C).
To evaluate the effects of CRT on cationic bovine serum albumin (C-BSA) induced MN mice, kidney index, levels of microalbumin from 24 h urine, creatinine, total cholesterol and serum albumin were detected after the routine induction of MN mice and drug treatment (Figure 2A). The microalbumin levels from 24 h urine were significantly increased in MN mice from the 4th week till the end of the experiment (p < .001, Figure 2B showed higher kidney index, higher levels of creatinine and total cholesterol, and lower level of serum albumin than that of normal mice (all p < .01), which were all significantly reversed by the treatment of CRT, especially in the CRT-H group (all p < .05), similar to that of prednisone ( Figure 2B,F). Immunohistochemical staining showed that MN led to a significant down-regulation of both nephrin and podocin proteins in MN mice compared with that in the normal group (both p < .001). In contrast, the integrity of nephrin and podocin distributions in the glomerular basement membrane (GBM) of MN mice were both rescued effectively at the 8th week following the treatment of CRT and prednisone (both p < .01, Figure 2 G,J,K). Pathologically, the GBM and focal segmental mesangial hypercellularity were observed in MN mice using periodic acid-silver-meth-enamine (PASM). Especially, GBM showed a slight thickening, as well as numerous spikes and bubbles ( Figure 2H). High dosage of CRT remarkedly improved the morphological changes of renal tissues in MN mice, similar to prednisone (p < .001, Figure 2 H,L). An increased granular deposition of IgG along the glomerular capillary walls was also observed in the renal tissues of MN mice compared with normal controls (p < .001, Figure 2I,M). MN mice in the CRT and prednisone treatment groups all showed lower levels of IgG deposition than that in the model group ( Figure 2I,M). These data suggest that CRT may effectively ameliorate the renal damage and immune injury caused by MN modeling.
Due to the prominent anti-inflammation, immuneregulation and hormone-like effects, 6,7 CRT has been authorized by National Medical Products Administration for treating rheumatoid arthritis and systemic lupus erythematosus. Our previous study revealed that CRT effectively ameliorated kidney functions and renal histopathology in diabetic kidney disease through recovering the balance of immune-inflammation system. 8 To verify its potentials against MN in a further level, we herein identified chemical and target profilings of CRT and investigated the 'disease genes-drug target' interaction network combined with the in vivo and in vitro experiment validations.
In conclusion, our findings identified CRT as a potential drug candidate for MN and revealed that the treatment of CRT may effectively ameliorate renal dysfunction and reduce the podocyte injury via targeting the TNF-α-IL-6-JAK2/STAT3-MMP9 signalling axis, which may facilitate its clinical application in MN therapy.

C O N F L I C T O F I N T E R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.